Detection of Bacteroides forsythus by immunomagnetic capture and a polymerase chain reaction-DNA probe assay.
The aim of this study was to combine immunomagnetic capture and a polymerase chain reaction (PCR) followed by hybridization with a DNA probe for the detection of Bacteroides forsythus. Magnetic beads were coated with the immunoglobulin G fraction of an antiserum specific for B. forsynthus. Aliquots were incubated with various concentrations of a suspension of B. forsythus or with a suspension containing 16 bacterial species, at a concentration of 10(10) cells/ml, spiked with dilutions of B. forsythus. Beads with bound bacteria were boiled, and the target DNA in the supernatant was amplified to generate a 392-bp PCR fragment specific for B. forsythus. The amplified product was detected by dot-blot hybridization with a digoxigenin-labeled 392-bp probe. The detection limit was determined to be 10 cells/ml using immunocapture on a suspension of B. forsythus and 100 on spiked bacterial suspensions. Subgingival plaque samples were obtained from 39 Bolivian individuals with poor oral hygiene. Each sample was analyzed by the above procedure and by immunofluorescence. The overall prevalence of individuals harboring B. forsythus was 62% by immunofluorescence and 82% by PCR-DNA probe assay. The immunocapture, PCR. DNA-probe procedure should be useful for the detection of B. forsythus, particularly in false-negative samples obtained by less sensitive techniques.
Chandad
F
F
Groupe de Recherche en Ecologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec, Canada.
Guillot
E
E
Mouton
C
C
D000293:Adolescent; D000328:Adult; D000368:Aged; D015373:Bacterial Typing Techniques; D001439:Bacteroides / Q000302:isolation & purification*; D002648:Child; D002675:Child, Preschool; D015342:DNA Probes; D003773:Dental Plaque / Q000382:microbiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D015342:DNA Probes